In last year's report, we described the lack of correlation between the kinetics of the bacteriorhodopsin (BR) photocycle measured by optical spectroscopy and by voltage generation across the energy-transducing membrane. We considered and evaluated two different explanations. 1.) Thermodynamic back-pressure directly decreased kinetic constants in the photocycle transitions. 2.) The apparent difference was explained by slow major electrogenic events that showed negligible optical changes. In this case, the effects of adding uncouplers would be due to their lowering of the electrical resistance (R) of the membrane and consequently the RC ( C for capacitance) constant leading to faster electrical response times. Our results, at the time, favored explanation 1. Because, of the importance of establishing the true explanation for the phenomenon, we repeated and extended the controls that had been performed earlier. The new data confirmed our earlier conclusions and the work was submitted for publication in The European Journal of Biochemistry and accepted. In order to achieve the original goal of correlating the kinetics of voltage generation with the optical kinetics of photocycle turnover to identify the energy-transducing steps, we turned to a new approach. The system we used to follow the kinetics of voltage generation consists of small samples of fixed membranes in a closed vesicular conformation. The voltage that builds causes the slowdown in kinetics. We have started to construct a new system which uses open membrane fragments electrically oriented in a gel. No standing voltage is formed, but with electrodes, one can measure the kinetics of current (i.e. pumped protons) movements. An additional advantage of this system is that both the optical and electrical responses can be measured on the same sample. Combining this approach with our new discovery of a specific kinetic model which describes the BR-photocycle (described in Z01 HL00401-35) could lead to the identification of the energy-transducing steps in the photocycle. The kinetic model involves two separate, but parallel photocycles, one with five steps, and the other with three. We believe that the 5-step cycle may be energy-transducing and that the 3-step cycle, energy-dissipating. In previous reports, we described experimental data which implicated the importance of a membrane squalene-phosphatidyl glycerophosphate interaction with a BR-peripheral aspartic acid residue for the energy-transducing function of the photocycle. To test this idea, we arranged with Dr. Martin Engelhard in Germany to prepare 15-C-aspartic acid-labeled-BR. In collaboration with Dr. Robert Tycko of NIDDK, we examined the solid state NMR signals from this preparation and compared them with a Triton X-100-treated preparation, which is known to destroy the suspected energy-transducing part of the photocycle. The results indicated a subtle perturbation in signals that have not been assigned to any of the internal aspartic acid residues. Therefore, these may be due to the peripheral residues. We plan to repeat these experiments and to find if reconstitution of the Triton-damaged preparation with extracted membrane lipids, under conditions known to restore native behavior to the membranes, restores the native pattern for signals from the labeled aspartate residues.